The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Estimation of potential risk of allyl alcohol induced liver injury in diabetic patients using type 2 diabetes spontaneously diabetic Torii-Leprfa (SDT fatty) rats
Tadakazu TakahashiChizuru MatsuuraKaoru ToyodaYusuke SuzukiNaohito YamadaAkio KobayashiShoichiro SugaiKayoko Shimoi
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2019 Volume 44 Issue 11 Pages 759-776

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Abstract

In order to estimate the potential risk of chemicals including drug in patients with type 2 diabetes mellitus (T2DM), we investigated allyl alcohol induced liver injury using SD rats and Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats as a model for human T2DM. The diabetic state is one of the risk factors for chemically induced liver injury because of lower levels of glutathione for detoxification by conjugation with chemicals and environmental pollutants and their reactive metabolites. Allyl alcohol is metabolized to a highly reactive unsaturated aldehyde, acrolein, which is detoxified by conjugation with glutathione. Therefore, we used allyl alcohol as a model compound. Our investigations showed that SDT fatty rats appropriately mimic the diabetic state in humans. The profiles of glucose metabolism, hepatic function tests and glutathione synthesis in the SDT fatty rats were similar to those in patients with T2DM. Five-week oral dosing with allyl alcohol to the SDT fatty rats revealed that the allyl alcohol induced liver injury was markedly enhanced in the SDT fatty rats when compared with the SD rats and the difference was considered to be due to lower hepatic detoxification of acrolein, the reactive metabolite of allyl alcohol, by depleted hepatic glutathione synthesis. Taking all the results of the present study into consideration, the potential for allyl alcohol to induce liver injury is considered to be higher in diabetic patients than in healthy humans.

INTRODUCTION

Allyl alcohol is used not only as a raw material for the production of glycerol but also as a precursor to many specialized compounds such as flame-resistant materials, drying oils and plasticizers (Krähling et al., 2002). Allyl alcohol is metabolized to a reactive metabolite, acrolein, by alcohol dehydrogenase (ADH), and this is detoxified by conjugation with glutathione and excreted in the urine as mercapturic acid metabolites (Serafini-Cessi, 1972; Patel et al., 1983; Stevens and Maier, 2008). Acrolein, a highly reactive unsaturated aldehyde, is also a ubiquitous environmental pollutant and its potential as a serious environmental health threat is beginning to be recognized (Moghe et al., 2015). Humans are exposed to acrolein by the oral (food and water), respiratory (automobile exhaust, and biocide use) and dermal routes, in addition to endogenous generation (metabolism and lipid peroxidation) (Moghe et al., 2015). Acrolein has been suggested to play a role in several disease states including spinal cord injury, multiple sclerosis, Alzheimer’s disease, cardiovascular disease, diabetes mellitus, and neuro-, hepato-, and nephro-toxicity (Moghe et al., 2015). At the cellular level, acrolein exposure has diverse toxic effects, including DNA and protein adduction, oxidative stress, mitochondrial disruption, membrane damage, endoplasmic reticulum stress, and immune dysfunction (Moghe et al., 2015).

Glutathione is the most abundant cellular thiol antioxidant and exhibits numerous and versatile functions. Disturbances in glutathione (GSH) homeostasis have been associated with liver disease induced by drugs, alcohol, food and environmental pollutants (Chen et al., 2013). Lower levels of glutathione have been reported to be a potential risk of drug induced liver injury (DILI) related to acetaminophen in humans (Sun et al., 2009) and this is reasonable because reactive metabolites, which are often the cause of DILI, are detoxified by conjugation with glutathione. Decreased levels of glutathione have been observed in the blood of diabetic patients possibly due to compromised levels of GSH synthesis and metabolism enzymes (Illing et al., 1951; Lal and Kumar, 1967; Awadallah et al., 1978; Lagman et al., 2015). In addition, the diabetic state has been regarded as one of the risk factors of DILI (Chalasani et al., 2014; Gaude et al., 2015; Ortega-Alonso et al., 2016; Lu et al., 2016) and this consideration could be applicable not only to drugs but also to other exogenous substances which are detoxified by glutathione conjugation. In other words, the potential for liver injury induced by chemicals or environmental pollutants is considered to be higher in diabetic patients than in healthy humans. Thus, it is important to estimate the risk of liver injury induced by chemicals and environmental pollutants in diabetic patients.

In order to investigate the risk of liver injury from allyl alcohol in the diabetic patients, we used Spontaneously Diabetic Torii-Leprfa (SDT fatty) rats as an animal model of human diabetes. The SDT fatty rat is a new model of obese type 2 diabetes, established by introducing the fa allele of the Zucker fatty rat into the Spontaneously Diabetic Torii (SDT) rat genome (Shinohara et al., 2000; Masuyama et al., 2005). The SDT fatty rats show overt obesity and hyperglycemia and hyperlipidemia from a young age (from four to six weeks of age) (Matsui et al., 2008). The pathology of diabetic complications in the SDT fatty rats has been reported to include diabetic nephropathy, cataracts, retinal findings and osteoporosis (Matsui et al., 2008; Katsuda et al., 2014). In the present study, we investigated the hepatotoxicity of allyl alcohol in the SDT fatty rats comparing with intact SDT fatty rats. We also investigated the similarity of the profiles of glucose metabolism, hepatic function tests and glutathione synthesis between the SDT fatty rats and diabetic patients to validate the usefulness of the SDT fatty rats as the animal model for human type 2 diabetes mellitus (T2DM).

MATERIALS AND METHODS

Animals

Fifteen male Jcl:SD (SD) rats and thirteen male SDT fatty rats at 12 and 5 weeks of age, respectively, were obtained from CLEA Japan Inc. (Tokyo, Japan). The animals were housed individually in wire-mesh cages kept in an air-conditioned room with a 12-hr light-dark cycle (lighting from 7:00 a.m. to 7:00 p.m.) at a temperature of 23 ± 1°C, a relative humidity of 55 ± 5% and a ventilation rate of about 15 times per hour. The SD and SDT fatty rats were quarantined and acclimated for 1 and 8 weeks, respectively, and were allowed free access to a commercial pelleted diet (CRF-1, Oriental Yeast Co., Ltd., Tokyo, Japan) ad libitum during the quarantine/acclimated period. Tap water was available for drinking ad libitum. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc. This study was conducted in accordance with the Japanese Law for the Humane Treatment and Management of Animals (Law No. 105, as revised in 2013, issued in October 1, 1973).

Dosing of allyl alcohol

Allyl alcohol was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and was diluted with water for injection (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan). Each strain of rats was randomly allocated to each group based on the body weights measured on the day before the initiation of dosing so that the initial mean body weights of each group were equivalent. Allyl alcohol was administered orally once daily to the SD and SDT fatty rats for 5 weeks (13 to 18 weeks of age, 4 or 5 rats of each strain per group). The dose levels of 10 and 30 mg/kg was selected for both the SD and SDT fatty rats as the dose level expected for the effects on the liver (increases in the plasma transaminase activities and hepatocellular damage) based on the results from a 13-week gavage toxicity study of allyl alcohol in Fischer 344 rats and B6C3F1 mice (National Toxicology Program, 1995). The animals in the matched control groups for each strain of rats were given the vehicle (water for injection). The dosing volume was set at 5.0 mL/kg and was calculated individually based on the most recently recorded body weight during the dosing period.

Observations, measurements and examinations

Clinical observations, measurements of body weights, food consumption, food efficiency and water intake

The animals were observed carefully for any clinical signs twice daily (before and after dosing) during the dosing period. The animals were weighed and the food consumption per animal was calculated at frequent intervals from the initiation of dosing (day 1) to the end of the dosing (day 34). Food efficiency during the dosing period was calculated individually. Daily water intake during the urine sampling (on day 30) was measured for all the animals except for the SDT fatty rats at 30 mg/kg.

Urine, blood and liver sampling

Urine samples were collected from the SD and SDT fatty rats in the control and allyl alcohol treated groups on day 30 (age of the animals: 17 weeks) of the dosing period as described previously (Kondo et al., 2012) for the measurement of urine volume per day and for determination of glucose (GLU), creatinine (CRN), creatine (CR) and taurine (TAU). However, urine samples were not collected from the SDT fatty rats at 30 mg/kg because of the early sacrifice of the animals (up to day 29).

Sequential blood sampling was performed for all the SD and SDT fatty rats in the control and allyl alcohol treated groups under the fed condition between 8:00 a.m. and 10:00 a.m. during the pre-dosing period (on day -5) and on days 14 and 34 of the dosing period (age of the animals: 12, 14 and 17 weeks). However, blood samples on day 34 were not collected from the SDT fatty rats at 30 mg/kg because of the early sacrifice of the animals (up to day 29). Blood samples were collected from the subclavian vein into lithium heparin-treated syringes without anesthesia. Plasma collection for the measurement of the following parameters were conducted as described previously (Kondo et al., 2012): aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltranspeptidase (GGT), lactate dehydrogenase (LDH), glutamate dehydrogenase (GLDH), sorbitol dehydrogenase (SDH) and guanase (GU).

At the end of the dosing period (on day 36, age of the animals: 18 weeks), the animals’ abdomens were opened under isoflurane anesthesia and blood samples were taken from the abdominal aorta under the fed condition. Plasma samples, obtained as described previously (Kondo et al., 2012), were used for the measurements of plasma GLU concentrations, plasma parameters for glutathione-related endogenous metabolite (creatinine (CRN), creatine (CR), lactic acid (LA) and pyruvic acid (PA)), and plasma parameters related to the hepatobiliary system (alkaline phosphatase (ALP), total bilirubin (T-BIL), direct bilirubin (D-BIL) and total bile acid (TBA)). Then, samples of the liver were taken from all the SD and SDT fatty rats in the control and allyl alcohol treated groups and frozen with liquid nitrogen. The liver samples were stored frozen in an ultra-deep freezer set at –80°C until use. However, for the SDT fatty rats at 30 mg/kg, the procedure described above were conducted on days 22 and 29 (age of the animals: 17 weeks).

Measurements of urinary and plasma parameters for glucose metabolism and glutathione-related endogenous metabolites and hepatic function

Urinary and plasma GLU concentrations were measured at 37°C with a TBA-120FR automated analyzer (Toshiba, Tokyo, Japan) using standard reagents by the HX-G-6-PDH method (FUJIFILM Wako Pure Chemical Corporation). Plasma LA and PA concentrations were measured at 37°C with a TBA-120FR automated analyzer (Toshiba Corporation) using standard reagents by the lactate oxidase·peroxidase enzyme method (Kyowa Medex Co., Ltd., Tokyo, Japan) and the pyruvate oxidase·peroxidase enzyme method (Kyowa Medex Co., Ltd.), respectively. Urinary CR, CRN and TAU concentrations and plasma CR and CRN concentrations were determined by liquid chromatography-mass spectrometry (LC/MS): API 4000 QTRAP (AB SCIEX, Inc., Tokyo, Japan) equipped with a Prominence series (Shimadzu, Kyoto, Japan) after deproteination. Creatine-d3 and 2-aminoethane-d4-sulfonic acid (Medical isotopes INC., Pelham, NH, USA) and Creatinine-d3 (CDN isotopes, pointe-Claire, Quebec, Canada) were used as the internal standards for CR, TAU and CRN, respectively. For the urinary endogenous metabolites, the excretion per day was calculated for each metabolite by multiplying the urine volumes and concentrations.

Plasma AST, ALT, LDH, GLDH, SDH, GU and ALP activities were measured at 37°C with a TBA-120FR automated analyzer using standard reagents by the UV kinetic method for AST, ALT, LDH and ALP (Wako Pure Chemical), GLDH (RANDOX Laboratories Ltd., Antrim, UK), SDH (SEKISUI MEDICAL CO., Ltd., Tokyo, Japan) and GU (Serotec Co., Ltd., Hokkaido, Japan). Plasma GGT activities, plasma T-BIL, D-BIL and TBA concentrations were measured at 37°C with a TBA-120FR automated analyzer using standard reagents by the enzymatic method for these parameters (GGT: Wako Pure Chemical, T-BIL and D-BIL: KANTO CHEMICAL CO., INC., Tokyo, Japan, TBA: KAINOS Laboratories, Inc., Tokyo, Japan).

Measurements of the weights of the liver, carcass of the animals and white adipose tissue in the abdominal cavity

At the terminal sacrifice (age of the animals: 17 or 18 weeks), the final body weights from all the animals were recorded to calculate the relative weights of the liver, carcass of the animals and white adipose tissue in the abdominal cavity to the body weights. The liver, carcass of the animals and white adipose tissue in the abdominal cavity were weighed and the relative weights of each organ to the final body weights were calculated.

Assay of hepatic levels of reduced glutathione, oxidized glutathione and lipid peroxide

Hepatic reduced-form (GSH) and oxidized-form (GSSG) glutathione levels were determined by an enzyme recycling method using a GSH/GSSG quantification kit (Dojindo Laboratories, Kumamoto, Japan) as described previously (Kondo et al., 2012).

The frozen liver samples were thawed and PBS (TAKARA BIO INC., Shiga, Japan) containing 0.1 wt% Triton X-100 (F. Hoffmann-La Roche, Ltd., Basel, Switzerland) and 0.05 wt% sodium deoxycholate (Wako Pure Chemical) was added to the samples at a volume of 4 mL per 1 g wet tissue weight. The tissue samples were homogenized using a Tissuelyser System (QIAGEN, Tokyo, Japan). The lipid peroxide (LPO) was extracted by shaking for 15 min at 4°C under protection from light. The homogenates were centrifuged at 3,000 g for 10 min at 4°C and the supernatants were obtained for measurement of LPO concentrations. Hepatic LPO concentrations in the supernatants were measured by a colorimetric method using an LPO-CC kit (KAMIYA BIOMEDICAL COMPANY, Seattle, WA, USA).

Measurements of mRNA of fatty acid beta-oxidation-related enzymes and gluconeogenesis-related enzymes

Aliquots of the tissue samples were homogenized by a Tissue Lyser (QIAGEN) and the total RNA was extracted using an RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. 2.0 μg of the isolated total RNA was used to synthesize cDNA with SuperScript VILO Mastermix (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The synthesized cDNA solutions were diluted 5-fold with Tris-EDTA (TE) buffer (pH8.0, NIPPON GENE CO., LTD., Tokyo, Japan). Before the measurements, the cDNA solutions were further diluted 10-fold with MILLI-Q water (Merck Millipore, Burlington, MA, USA) and were used for TAQman probe-based semi quantitative-real time PCR. The mRNA levels were measured in duplicate on a QuantStudioTM3 Real-Time PCR System (Thermo Fischer Scientific) using TAQman Gene Expression Master Mix (Thermo Fisher Scientific) according to the manufacturer’s instructions. The data analysis was performed by a calibration curve method using SDS software (Thermo Fisher Scientific) and the results were normalized to ACTB expressions. The following primer and TAQman probe mixtures were obtained from Thermo Fisher Scientific: carnitine palmitoyltransferase 1A (Cpt1a, Rn00580702_m1), acyl-CoA dehydrogenase medium chain (Acadm, Rn00566390_m1), pyruvate carboxylase (Pc, Rn00562534_m1), pyruvate kinase L/R (Pklr, Rn01455286_m1), phosphoenolpyruvate carboxykinase 1 (Pck1, Rn01529014_m1), glucose-6-phosphatase, catalytic subunit (G6pc, Rn00689876_m1), fructose-bisphosphatase 1 (Fbp1, Rn00561189_m1) and actin, beta (Actb, Rn00667869_m1).

Histopathological examination of the liver

The left lobe of the liver was cut into longitudinal sections and the liver slices were embedded in paraffin. Sectioning and hematoxylin-eosin staining was performed according to routine histological procedures.

Statistical analysis

The mean values and standard deviations in each group were calculated for the body weights, food consumption, food efficiency, water intake and urine volume, urine or plasma parameters for glucose metabolism, glutathione-related endogenous metabolites and hepatic function, the weights of the liver, carcass of the animals and white adipose tissue in the abdominal cavity, hepatic GSH and GSSG concentrations, hepatic LPO concentrations and mRNA levels of fatty acid β-oxidation-, glycolysis- and gluconeogenesis-related enzymes during and/or at the end of the dosing period. A Student’s t test was conducted for comparison of the parameters mentioned above between the control SD and SDT fatty rats and between the vehicle control and allyl alcohol treated groups each of the SD and SDT fatty rats (except for the data obtained from the 30 mg/kg group in the SDT fatty rats at the end of the dosing period because of the early sacrifice of the animals). The levels of significance were set at 5% and 1% (one- or two-tailed).

RESULTS

Characteristics of glucose metabolism, hepatic function and glutathione synthesis in the intact SDT fatty rats

To investigate how much the SDT fatty rats appropriately mimic the diabetic state in humans, the profiles of glucose metabolism, hepatic function tests and glutathione synthesis were compared between the control SD and SDT fatty rats.

For glucose-related parameters, the SDT fatty rats had markedly higher levels in water intake, urine volume, urinary and plasma GLU (Table 1) (p < 0.01).

Table 1. Comparison of the control parameters in the SD and SDT fatty rats.
Parameters (unit) Weeks
of age
Strain
SD SDT fatty
Water intake (mL/day) 17 57.84 ± 16.72 273.55 ± 27.92 ##
Urine volume (mL/day) 17 41.42 ± 17.16 280.95 ± 35.13 ##
Urinary GLU excretion (mg/day) 17 1.532 ± 1.364 18164.970 ± 880.057 ##
Plasma GLU levels (mg/dL) 18 246.6 ± 13.2 751.0 ± 26.2 ##
Cumulative body weight gain (g) 13 to 18 119.44 ± 16.34 -6.15 ± 9.14 ##
Cumulative food consumption (g) 13 to 18 975.38 ± 74.58 1997.10 ± 106.38 ##
Food efficiency 13 to 18 12.20 ± 0.87 -0.33 ± 0.46 ##
Carcass weights (g) 18 383.70 ± 14.93 242.48 ± 13.08 ##
Carcass weights (g/100g BW) 18 70.54 ± 2.47 49.75 ± 1.72 ##
White adipose tissue weights (g) 18 14.750 ± 3.928 44.850 ± 1.756 ##
White adipose tissue weights (g/100g BW) 18 2.692 ± 0.652 9.208 ± 0.352 ##

Abbreviation: GLU; glucose, Carcass; carcass of the animals

All the parameters were measured for each rat (n = 4 or 5/group/point) at the weeks of age shown in the table. Data are shown as mean ± SD. ## p<0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test)

For growth-related parameters, the SDT fatty rats had higher values of cumulative food consumption per animal and lower values of cumulative body weight gain and food efficiency from 13 to 18 weeks of age (Fig. 1 and Table 1) (p < 0.01), lower weights of carcass of the animals and higher weights of the white adipose tissue in the abdominal cavity at 18 weeks of age (Table 1) (p < 0.01).

Fig. 1

Body weights and food consumption in the allyl alcohol treated SD and SDT fatty rats during the dosing period. In the allyl alcohol treated SDT fatty rats, all the animals at 30 mg/kg were sacrificed by day 29 due to the deterioration of the animals’ physical condition. Mean values are shown in the figure. # p < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

For hepatic mRNA of fatty acid β-oxidation-, glycolysis- and gluconeogenesis-related enzymes, the SDT fatty rats had higher hepatic Cpt1a levels, slightly lower hepatic Acadm levels, higher hepatic Fbp1 levels, and lower hepatic Pklr and G6pc levels at 18 weeks of age (Fig. 2) (p < 0.01 or p < 0.05).

Fig. 2

mRNA of fatty acid beta-oxidation-, glycolysis- and gluconeogenesis-related enzymes in the liver in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: Cpt1a; carnitine palmitoyltransferase 1A, Acadm; acyl-CoA dehydrogenase medium chain, Pc; pyruvate carboxylase, Pklr; pyruvate kinase L/R, Pck1; phosphoenolpyruvate carboxykinase 1, G6pc; glucose-6-phosphatase, catalytic subunit, Fbp1; fructose-bisphosphatase 1, Actb; actin, beta. The liver samples were obtained from each rat at the end of the dosing period (day 36) (n = 4 or 5/group) under non-fasted conditions. The liver samples at 30 mg/kg in the SDT fatty rats were collected from each rat until day 29 because of the early sacrifice due to the deterioration of the animals’ physical condition. # < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

For plasma glutathione-related endogenous metabolites, the SDT fatty rats had higher urinary CR levels, lower urinary and plasma CRN levels, lower urinary TAU levels, higher plasma PA levels (Figs. 3 and 4) (p < 0.01 or p < 0.05) and no changes in plasma LA levels at 18 weeks of age (Fig. 4). Plasma CR levels were higher in the SDT fatty rats than those in the SD rats at 13 and 15 weeks of age but the change disappeared up to 18 weeks of age (Fig. 3).

Fig. 3

Plasma CR and CRN levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: CR; creatine, CRN; creatinine. Blood samples were collected at 9:00-10:00 a.m. from each rat on the days shown in the figure (n = 4 or 5/group/point) under non-fasted conditions. “Pre”, “14D” and “34D” indicates day -5 in the pre-dosing period and days 14 and 34 in the dosing period, respectively. In the allyl alcohol treated SDT fatty rats, all the animals at 30 mg/kg were sacrificed by day 29 due to the deterioration of the animals’ physical condition. Individual data are shown in the figure. # p < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

Fig. 4

Urinary CR, CRN and TAU levels and plasma PA and LA levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: U-CR; urinary creatine excretion, U-CRN; urinary creatinine excretion, U-TAU; urinary taurine excretion, P-PA; plasma pyruvic acid levels, P-LA; plasma lactic acid levels. Urine samples were collected approximately 24 hr from each rat on day 30 (n = 4 or 5/group) under non-fasted conditions except for the 30 mg/kg group in the SDT fatty rats. Blood samples were collected at 9:00-10:00 a.m. from each rat at the end of the dosing period (day 36) (n = 4 or 5/group) under non-fasted conditions. Blood samples at 30 mg/kg in the SDT fatty rats were collected from each rat until day 29 because of the early sacrifice due to the deterioration of the animals’ physical condition. Data are shown as mean ± standard deviation. # p < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

For the hepatic glutathione and oxidative stress levels, the SDT fatty rats had higher values of the liver weights, lower hepatic GSH and GSSG levels and higher LPO levels at 18 weeks of age (Fig. 5 and Table 2) (p < 0.01).

Fig. 5

Hepatic GSH, GSSG and LPO levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: GSH; reduced glutathione, GSSG; oxidized glutathione, LPO; lipid peroxide. The liver samples were obtained from each rat at the end of the dosing period (day 36) (n = 4 or 5/group) under non-fasted conditions. The liver samples at 30 mg/kg in the SDT fatty rats were collected from each rat until day 29 because of the early sacrifice due to the deterioration of the animals’ physical condition. Data are shown as mean ± standard deviation. ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

Table 2. The liver weights in the allyl alcohol treated SD and SDT fatty rats.
Strain: SD rats SDT fatty rats
Dose levels (mg/kg): 0 10 30 0 10 30
Absolute weights (g) 18.254 ± 1.194 18.174 ± 2.454 19.288 ± 0.912 22.950 ± 1.924 ## 23.265 ± 1.824 27.484 ± 7.776
Relative weights (g/100g B.W.) 3.356 ± 0.196 3.240 ± 0.122 3.550 ± 0.308 4.703 ± 0.231 ## 4.720 ± 0.225 5.870 ± 1.589

The liver weights were measured at the end of the dosing period (on day 36) under non-fasted conditions (n = 4 or 5/group). The liver weights at 30 mg/kg in the SDT fatty rats were measured for each rat until day 29. Data are shown as mean ± S.D. ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05 significantly different from the matched control group (Student’s t-test).

For the hepatic function parameters, the SDT fatty rats had higher plasma levels of AST, ALT, GGT, GLDH, SDH, ALP and D-BIL at 18 weeks of age (Figs. 6, 7 and 8) (p < 0.01). The SDT fatty rats had higher plasma TBA levels (Fig. 8) (p < 0.05) and no increases in plasma LDH or GU levels at 18 weeks of age (Figs. 6 and 7).

Fig. 6

Plasma AST, ALT, GGT and LDH levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: AST; aspartate aminotransferase, ALT; alanine aminotransferase, GGT; gamma-glutamyltranspeptidase, LDH; lactate dehydrogenase. Blood samples were collected at 9:00-10:00 a.m. from each rat on the days shown in the figure (n = 4 or 5/group/point) under non-fasted conditions. “Pre”, “14D” and “34D” indicates day -5 in the pre-dosing period and days 14 and 34 in the dosing period, respectively. In the allyl alcohol treated SDT fatty rats, all the animals at 30 mg/kg were sacrificed by day 29 due to the deterioration of the animals’ physical condition. Individual data are shown in the figure. # p < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

Fig. 7

Plasma GLDH, SDH and GU levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: GLDH; glutamate dehydrogenase, SDH; sorbitol dehydrogenase, GU; guanase. Blood samples were collected at 9:00-10:00 a.m. from each rat on the days shown in the figure (n = 4 or 5/group/point) under non-fasted conditions. “Pre”, “14D” and “34D” indicates day -5 in the pre-dosing period and days 14 and 34 in the dosing period, respectively. In the allyl alcohol treated SDT fatty rats, all the animals at 30 mg/kg were sacrificed by day 29 due to the deterioration of the animals’ physical condition. Individual data are shown in the figure. ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

Fig. 8

Plasma ALP, TBA, T-BIL and D-BIL levels in the allyl alcohol treated SD and SDT fatty rats. Abbreviation: ALP; alkaline phosphatase, TBA; total bile acid, T-BIL; total bilirubin, D-BIL; direct bilirubin. Blood samples were collected at 9:00-10:00 a.m. from each rat at the end of the dosing period (day 36) (n = 4 or 5/group) under non-fasted conditions. Blood samples at 30 mg/kg in the SDT fatty rats were collected from each rat until day 29 because of the early sacrifice due to the deterioration of the animals’ physical condition. Data are shown as mean ± standard deviation. # p < 0.05, ## < 0.01 significantly different between the control SD and SDT fatty rats (Student’s t-test), * p < 0.05, ** p < 0.01 significantly different from the matched control group (Student’s t-test).

Differences of the effects of allyl alcohol on the liver between the SD and SDT fatty rats

To estimate the potential risk of allyl alcohol induced liver injury in diabetic patients, the effects of allyl alcohol on the liver were compared between the SD and SDT fatty rats during and after repeated dosing of allyl alcohol for 5 weeks.

In the allyl alcohol treated SD rats, there were no treatment-related deaths or deterioration of the animals’ physical condition in any animal at either dose level. On the other hand, in the allyl alcohol treated SDT fatty rats, one animal at 30 mg/kg was sacrificed on day 22 and the other four animals at this dose level were sacrificed on day 29 due to the deterioration of the animals’ physical condition without apparent decreases in body weights or food consumption (Fig. 1). There were no treatment-related deaths or deterioration of the animals’ physical condition in any SDT fatty rat at 10 mg/kg.

In the allyl alcohol treated SD rats, the following changes were noted in plasma parameters for hepatic function and hepatobiliary system when compared with those in the control SD rats: slightly increased plasma AST and GLDH levels at 30 mg/kg on day 34 (Figs. 6 and 7) (p < 0.05), slightly decreased plasma GU levels at 10 mg/kg on days 14 and 34 (Fig. 7) (p < 0.05 or p < 0.01), increased plasma T-BIL levels at 30 mg/kg at the end of the dosing period (Fig. 8) (p < 0.05). The following treatment-related histopathological findings were observed in the liver in one or two allyl alcohol treated SD rats at both dose levels at the end of the dosing period (Table 3): very slight periportal degeneration/single cell necrosis of the hepatocytes, very slight periportal inflammatory cell infiltration, very slight to slight periportal brown pigment deposition in Kupffer cells, proliferation of the bile duct and periportal fibrosis.

Table 3. The histopathological findings in the liver in the allyl alcohol treated SD and SDT fatty rats.

In the allyl alcohol treated SDT fatty rats, the following changes were noted: markedly increased plasma levels of AST, ALT, GGT and GLDH in one animal at 10 mg/kg on days 14 and 34 and two animals at 30 mg/kg on day 14 (Figs. 6 and 7) (no statistical significance), increased plasma SDH levels at 10 mg/kg on day 34 (p < 0.05) and two animals at 30 mg/kg on day 14 (Fig. 7), markedly increased plasma LDH and GU levels in one animal at 30 mg/kg on day 14 (Figs. 6 and 7) (no statistical significance), markedly increased plasma levels of ALP, TBA, T-BIL and D-BIL in one animal at 30 mg/kg at the early sacrifice (Fig. 8) (not statistically analyzed). The absolute and relative liver weights were markedly increased only in two allyl alcohol treated SDT fatty rats at 30 mg/kg at the early sacrifice (Table 2) (not statistically analyzed). The following treatment-related histopathological findings were observed in the liver in the allyl alcohol treated SDT fatty rats at both dose levels at the end of the dosing period or at the early sacrifice (Fig. 9 and Table 3): moderate to severe periportal necrosis of the hepatocytes, very slight to severe periportal degeneration/single cell necrosis of the hepatocytes, very slight to moderate periportal inflammatory cell infiltration, very slight periportal accumulation of foamy cells, very slight to moderate periportal brown pigment deposition in Kupffer cells/hepatocytes, very slight to moderate proliferation of the bile ducts and periportal fibrosis.

Fig. 9

Light micrographs of the liver sections. Light micrographs of hematoxylin-eosin stained liver sections from SD rat in the control group (A), SD rat in the allyl alcohol treated group (B), SDT fatty rat in the control group (C) and SDT fatty rat in the allyl alcohol treated group (D). In the allyl alcohol treated SDT fatty rat, necrosis of the periportal hepatocytes (*), bile duct proliferation and fibrosis (black arrows) were observed.

The magnitude of the changes in the plasma hepatic function parameters were greater in the allyl alcohol treated SDT fatty rats than those in the SD rats (Figs. 6, 7 and 8). The onset of the changes in the plasma hepatic function parameters was also earlier in the allyl alcohol treated SDT fatty rats than that in the SD rats (Figs. 6 and 7). The dose level causing the changes in these plasma parameters was lower in the allyl alcohol treated SDT fatty rats than that in the SD rats (Figs. 6 and 7). The incidence and severity of the treatment-related histopathological findings suggestive of liver injury were markedly increased in the SDT fatty rats than those in the SD rats (Fig. 9 and Table 3).

In the allyl alcohol treated SD rats, the following changes were noted in urinary and plasma parameters for glutathione-related endogenous metabolites when compared with the control SD rats: higher tendency of plasma LA and PA levels at 30 mg/kg at the end of the dosing period (Fig. 4). In the allyl alcohol treated SDT fatty rats, the following changes were noted: increased plasma CR levels at 10 mg/kg on day 34 and at 30 mg/kg on day 14 (Fig. 3) (p < 0.05), decreased plasma LA levels at 30 mg/kg at the early sacrifice (Fig. 4) (not statistically analyzed), slightly increased plasma PA levels at 10 mg/kg at the end of the dosing period (p < 0.05) and decreased plasma PA levels at 30 mg/kg at the early sacrifice (Fig. 4) (not statistically analyzed). In comparison of the changes between the allyl alcohol treated SD and SDT fatty rats, the direction of the changes in plasma LA and PA levels was different between these rats at the same dose level (Fig. 4).

Hepatic GSH and GSSG levels in the allyl alcohol treated SD rats were increased at both dose levels at the end of the dosing period (p < 0.01) but not in the hepatic GSSG levels (Fig. 5). Hepatic LPO levels in the allyl alcohol treated SD rats were increased at 10 mg/kg at the end of the dosing period (Fig. 5) (p < 0.05). In the allyl alcohol treated SDT fatty rats, hepatic GSH and GSSG levels tended to be lower or were decreased at both dose levels at the end of the dosing period or at the early sacrifice (Fig. 5) (p < 0.05 at 10 mg/kg and not statistically analyzed at 30 mg/kg). Hepatic LPO levels in the allyl alcohol treated SDT fatty rats were comparable to those in the matched control group at the end of the dosing period or at the early sacrifice (Fig. 5).

Only in the allyl alcohol treated SDT fatty rats, hepatic mRNA levels of Pc, Pck1, Fbp1, Cpt1a and Acadm tended to be increased at 30 mg/kg at the early sacrifice (Fig. 2) (not statistically analyzed).

DISCUSSION

In the present study, we investigated 1) the characteristics of the SDT fatty rats from 13 weeks of age (the initiation of the study) to 18 weeks of age (the end of the study) in terms of the similarity of glucose metabolism, hepatic function and glutathione synthesis to those in the patients with T2DM, and 2) the potential for allyl alcohol to induce liver injury in T2DM patients by using the SDT fatty rats.

Similarity of characteristics of glucose metabolism, hepatic function and glutathione synthesis between the control SDT fatty rats and T2DM patients

Urinary and plasma glucose levels were higher in the SDT fatty rats than in the SD rats. These indicate that the SDT fatty rats employed in this study were in a diabetic state at the initiation of the experiment. It has been reported that the plasma insulin levels in the SDT fatty rats increase (hyperinsulinemia) from 4 to 8 weeks of age and decrease after 16 weeks of age (Matsui et al., 2008). Thus, the SDT fatty rats employed in this study were considered to be in the condition of insulin insufficiency by the end of the dosing period. The SDT fatty rats also had higher cumulative food consumption and lower cumulative body weight gain and food efficiency, lower carcass weights of the animals and higher weights of white adipose tissue in the abdominal cavity than the SD rats. These alterations noted in the SDT fatty rats indicate that energy loss and/or impaired glucose uptake in the muscle occurred in the SDT fatty rats.

The SDT fatty rats had higher hepatic Fbp1 mRNA levels (gluconeogenesis-related enzyme), lower Pklr mRNA levels (glycolysis-related enzyme) and lower G6pc mRNA levels (glycolysis-related enzyme). These indicate that gluconeogenesis accelerated and glycolysis was decelerated in the SDT fatty rats. The SDT fatty rats had higher hepatic Cpt1a mRNA levels (fatty acid β-oxidation-related enzyme). The hepatic Acadm mRNA levels were slightly lower in the SDT fatty rats than in the SD rats. In the diabetic state, hepatic β-oxidation is accelerated to synthesize ketone bodies in the liver and ketone bodies are used for ATP production in the peripheral tissues in which decreased utilization of glucose (Horie et al., 1981; Asayama et al., 1999; Mahendran et al., 2013; Puchalska and Crawford, 2017). Thus, the higher hepatic Cpt1a mRNA levels in the SDT fatty rats indicates that fatty acid β-oxidation was also accelerated due to the diabetic state of the animals. The biological significance of the decreased hepatic Acadm mRNA levels in the SDT fatty rats was considered to be low because the change was slight. The higher hepatic LPO levels in the SDT fatty rats were considered to be related to accelerated hepatic β-oxidation because reactive oxygen species are generated in β-oxidation process (Rosca et al., 2012). It has been reported that hepatic gluconeogenesis are accelerated in T2DM patients (Gerich and Nurjhan, 1993) and that insulin insufficiency leads to a decrease in rates of glycolysis in the liver in diabetic patients (Guo et al., 2012). The profile of glucose metabolism in the SDT fatty rats was similar to that in the diabetic patients. Insulin resistance is a characteristics feature of both T2DM and obesity, and both obese and lean insulin-resistant individuals manifest multiple disturbances in free fatty acid metabolism (DeFronzo, 2004; Abdul-Ghani et al., 2008). Insulin insufficiency leads to acceleration of catabolism of lipids. The SDT fatty rats used in this study was considered to have insulin insufficient due to pancreatic dysfunction because serum insulin levels were decreased from 16 weeks of age in the SDT fatty rats (Matsui et al., 2008). Thus, the accelerated fatty acid β-oxidation in the SDT fatty rats were considered to be due to insulin insufficiency and the characteristics of lipid metabolism was similar to that in diabetic patients.

Hepatic GSH and GSSG levels were lower in the SDT fatty rats than in the SD rats. This indicates that the glutathione synthesis was complicated, and the pool of glutathione was lower than that in the healthy SD rats. It has been reported that patients with T2DM have lower levels of GSH due to compromised GSH synthesis and metabolism enzymes (Illing et al., 1951; Lal and Kumar, 1967; Awadallah et al., 1978; Lagman et al., 2015). The results obtained in the present study for the SDT fatty rats are consistent with T2DM patients. It has been reported that hepatic GSH/GSSG levels in the restricted-fed rats were lower than those in the ad libitum-fed rats and the difference was considered to be due to decreased supply of amino acids in the glutathione synthesis because amino acids were considered to be more preferentially used for the gluconeogenesis rather than for glutathione synthesis (Kondo et al., 2012). The urinary and plasma glucose levels in the SDT fatty rats were much higher than those in the healthy SD rats at 17 or 18 weeks of age. In a diabetic state, the liver produces glucose from muscle-derived alanine via the glucose-alanine cycle (Dhahbi et al., 1999; Felig, 1973; Chakrabarty and Leveille, 1968) and, in the SDT fatty rats, the plasma glucose levels were considered to be maintained at high levels mainly by hepatic gluconeogenesis using alanine supplied by the muscles (Garber et al., 1976; Chakrabarty and Leveille, 1968). The SDT rats are also known to show abnormal glucose tolerance and high glucose levels due to the accelerated gluconeogenesis (Masuyama et al., 2004; Morinaga et al., 2008). Thus, the lower hepatic glutathione levels in the SDT fatty rats was also considered to be related to more preferential use of amino acids for gluconeogenesis rather than for glutathione synthesis.

Plasma and urine CRN levels were lower in the SDT fatty rats than in the SD rats. CRN is synthesized from CR in the muscle and released into the blood. These changes were considered to be related to the decrease in glutathione production in the liver because CR is a by-product of S-adenosylmethionine in the metabolites associated with the transsulfuration pathway from methionine to glutathione (Timbrell et al., 1995a, 1995b; Schnackenberg et al., 2009). Urine CR levels were higher in the SDT fatty rats than in the SD rats. This change is considered not to reflect the production of hepatic glutathione. CR is supposed to be decreased if SAMe and its precursor, methionine, are decreased due to preferential use of aspartic acid, a precursor of methionine, in accelerated gluconeogenesis. On the other hand, it’s known that urinary excretion of CR is increased due to renal tubular dysfunction caused by mercury II chloride (renal cortical toxicant) (Waterfield et al., 1993). The possible explanation of the higher urinary CR levels in SDT fatty rats is increased urinary excretion of CR due to renal tubular dysfunction that developed spontaneously in the SDT fatty rats. The urinary TAU levels were lower in the SDT fatty rats than in the SD rats. This was considered to be related to the decreased hepatic glutathione synthesis because TAU is a by-product of cysteine. The higher plasma PA levels in the SDT fatty rats may be related to increased supply of alanine from the skeletal muscle due to accelerated gluconeogenesis.

Hepatic function parameters, plasma levels of AST, ALT, GGT, GLDH, SDH, ALP and D-BIL were higher in the SDT fatty rats than in the SD rats. It has been reported that the serum hepatic function parameters (AST, ALT, GGT, ALP and D-BIL) are higher in the diabetic patients than individuals who do not have diabetes (Doi et al., 2007; Dango et al., 2016; Mathur et al., 2016; Kanwar and Saxena, 2018; Roy et al., 2018).

The higher levels of hepatic function parameters in the SDT fatty rats were not accompanied by any histopathological changes suggestive of hepatotoxicity in the liver. These results indicate that the higher levels of hepatic function parameters in the SDT fatty rats are not related to hepatotoxicity. Transaminases are involved in the alanine-glucose cycle and the activity of transaminase is increased in the liver or small intestine when gluconeogenesis and protein catabolism are accelerated (Hagopian et al., 2003; Kobayashi et al., 2009, 2011). GLDH is also involved in the alanine-glucose cycle and may be increased when gluconeogenesis and protein catabolism are accelerated. From these observations, higher levels of plasma transaminases and GLDH in the SDT fatty rats are considered to be related to acceleration of gluconeogenesis and protein catabolism in this animal model. The absence of changes in plasma GU and LDH levels in the SDT fatty rats supported this discussion because plasma GU and LDH levels are increased in liver injury and not increased by enzyme protein synthesis like transaminases. The Hisayama study showed that serum GGT and ALT levels are strong predictors of diabetes in the general population, independent of known risk factors (Doi et al., 2007). The higher base line of the plasma GGT and ALT levels in the SDT fatty rats were consistent with the results of the Hisayama study. GGT plays key roles in GSH homeostasis by breaking down extracellular GSH and providing cysteine, the rate-limiting substrate, for intracellular de novo synthesis of GSH (Zhang et al., 2005). The higher base line of the plasma GGT levels in the SDT fatty rats were considered to be adaptive changes to maintain hepatic glutathione synthesis. It is well known that feeding increases serum ALP in rats and fasting reduces the serum ALP (Jackson, 1952; Young et al., 1981). Thus, the higher levels of plasma ALP in the SDT fatty rats may be related to increased food consumption. The higher levels of plasma D-BIL in the SDT fatty rats may be related to induction of the enzymes related to bilirubin synthesis (heme oxygenase-1) or bilirubin-conjugating enzymes due to increased oxidative stress (Kobayashi et al., 2003, Vítek, 2012). The higher levels of plasma TBA may be related to higher levels of plasma total cholesterol levels (data are not shown) suggestive of increased cholesterol synthesis in the liver in the SDT fatty rats. The plasma SDH levels were higher in the SDT fatty rats and there is a possibility that enzyme protein synthesis is increased due to altered glucose metabolism in the SDT fatty rats. Thus, the profile of changes in hepatic function tests in the SDT fatty rats was considered to be similar to that in the diabetic patients.

From these results, the characteristics of the SDT fatty rats is similar to that of T2DM patients in terms of glucose metabolism, liver function and hepatic glutathione synthesis.

Estimation of the potential risk of allyl alcohol induced liver injury in diabetic patients

In the SD rats, allyl alcohol induced hepatotoxicity was very slight based on the alteration of hepatic function parameters and histopathological changes in the liver. The allyl alcohol induced hepatotoxicity in the SD rats in the present study is similar to that in previously reported four-week oral dose study of allyl alcohol in SD rats in the Toxicogenomics Informatics Project conducted by the National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN, Japan). On the other hand, in the SDT fatty rats, allyl alcohol induced hepatotoxicity was more prominent based on comparison of the alteration of hepatic function parameters and histopathological changes in the liver between the SD and SDT fatty rats.

Allyl alcohol is metabolized to a reactive metabolite, acrolein, by ADH, and acrolein is detoxified by conjugation with glutathione (Serafini-Cessi, 1972; Patel et al., 1983; Stevens and Maier, 2008). The metabolic enzymes induced after repeated dosing of toxicants include those related to oxidative metabolism (CYPs) and those related to conjugation with glucuronic acid, sulfate, glycine, glutamine and glutathione. After repeated dosing of acetaminophen (APAP) to rats or mice, they become resistant to the APAP-induced hepatotoxicity (O’Brien et al., 2000; Buttar et al., 1976, 1977; Strubelt et al., 1979; Poulsen and Thomsen, 1988; Shayiq et al., 1999). The APAP-induced hepatotoxicity is caused by a reactive metabolite of APAP, N-Acetyl-4-benzoquinone imine (NAPQI) (Hodgman and Garrard, 2012). The increased hepatic glutathione levels enhance detoxification of NAPQI (O’Brien et al., 2000; Kondo et al., 2012). In the allyl alcohol treated SD rats, hepatic GSH levels were increased. The increased hepatic GSH levels were considered to be due to induction of metabolic enzymes related to detoxification of acrolein and the SD rats were considered to adapt themselves to allyl alcohol treatment. The higher tendency for the plasma LA and PA levels noted at 30 mg/kg in the allyl alcohol treated SD rats was considered to be related to the accelerated glutathione synthesis due to allyl alcohol treatment because LA and PA are byproducts for glutathione synthesis. On the other hand, in the SDT fatty rats, hepatic GSH and GSSG levels at both dose levels and plasma LA and PA levels at 30 mg/kg were lower than the control levels. These were considered to be related to the decrease in glutathione production in the liver. The endogenous metabolite profile of the allyl alcohol treated SDT fatty rats indicated that the endogenous metabolic capacity to detoxify allyl alcohol and its metabolites (acrolein) was lower in the SDT fatty rats than that in the SD rats. This was considered to be due to lower ability for hepatic glutathione synthesis caused by the preferential use of amino acids for accelerated gluconeogenesis rather than use of amino acids for glutathione synthesis under diabetic condition. From these observations, not only the base levels of hepatic glutathione synthesis but also the inducibility of hepatic glutathione synthesis after dosing allyl alcohol are lower in the SDT fatty rats than in the SD rats. The enhancement of allyl alcohol induced hepatotoxicity in the SDT fatty rats was considered to be related to lower ability of detoxification of acrolein due to lower hepatic GSH levels. In addition, in the SDT fatty rats, hepatic mRNA levels of Pc, Pck1 and Fbp1 (gluconeogenesis-related enzymes), and Cpt1a and Acadm (fatty acid β-oxidation-related enzymes) tended to be increased after treatment with allyl alcohol at 30 mg/kg. These were considered to be related to the deterioration of the animals’ physical conditions secondary to allyl alcohol induced hepatotoxicity.

In conclusion, the profiles of glucose metabolism, hepatic function tests and glutathione synthesis in the SDT fatty rats were similar to those in patients with T2DM. Taking all the results of the present study into consideration, the potential risk of allyl alcohol to induce liver injury is considered to be higher in diabetic patients than in healthy humans.

ACKNOWLEDGMENTS

The author would like to thank the invaluable contributions of Toshiyuki Shoda, Takuya Matsui, Kazuma Kondo, Yuki Tanaka, Koji Tada, Waka Shimizu, Masumi Takeda, Kazumi Ogawa, Eriko Sotomine, Risa Tsuchiya and the staff at the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, JAPAN TOBACCO INC.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
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